NOB was synthesized by an established method50 (link) and provided by the Ushio Chemix Corp. (Shizuoka, Japan). Geranic acid (85%), choline bicarbonate (~80% in water), dimethyl sulfoxide-d6 (99.5 atom %D), chloroform-d (99.8 atom % D), and phosphate buffered saline tablets were purchased from Merck KGaA (Darmstadt, Germany). Acetone, 4% paraformaldehyde phosphate buffer solution, formic acid, and ethanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Diethylene glycol ethyl methyl ether was purchased from Tokyo Chemical Industries (Tokyo, Japan). Porcine skin (Yucatan Micropig) was purchased from Charles River Laboratories International (Yokohama, Japan). All other chemicals were of the highest grade commercially available, and all solutions were prepared in deionized and distilled water.
Paraformaldehyde phosphate buffer solution
Manufactured by Fujifilm
Sourced in Japan, United States
Paraformaldehyde phosphate buffer solution is a chemical compound used as a fixative in various laboratory applications. It is a clear, colorless liquid that helps preserve the structure and morphology of biological samples, such as cells and tissues, for further analysis and examination.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8, by Leica (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Bz x710, by Keyence (3 mentions)
Oct compound, by Sakura Finetek (3 mentions)
Ethanol, by Fujifilm (2 mentions)
Matrigel, by BD (2 mentions)
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ckx41, by Olympus (2 mentions)
Fluoview fv10i, by Olympus (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Formalin neutral buffer solution, by Fujifilm (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Cryostat, by Leica (2 mentions)
Bz x700, by Keyence (2 mentions)
Histofine simple stain max po, by Nichirei Biosciences (2 mentions)
Alexa fluor 488 goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Bz x710 microscope, by Keyence (2 mentions)
89 protocols using paraformaldehyde phosphate buffer solution
1
Nonenolide Synthesis and Characterization
2
Endothelial Progenitor Cell Labeling
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PbMNCs and PbMNCs‐QQ were suspended in EGM‐2MV medium containing 5% FBS and depleted hydrocortisone (1 × 106 cells/mL), and were cultured in a human fibronectin‐coated 96‐well Primaria plate (1 × 105 cells/100 μL/well) for 7 days. On day 7, the medium was replaced with fresh medium containing FITC‐labeled Ulex europaeus agglutinin I (FITC‐UEA‐I, 10 μg/mL, Vector Lab., Burlingame, California) and 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindo‐carbocyanine perchlorate‐labeled acetylated low‐density lipoprotein (DiI‐acLDL, 10 μg/mL, Biomedical Tech., Stoughton, Massachusetts). The attached cells were cultured for 4 hours for EPC labeling. The cells were washed with PBS twice and fixed with 4% paraformaldehyde phosphate buffer solution (PFA, Wako, Osaka, Japan) at 4°C for 30 minutes. The wells were then covered by Vector shield with DAPI (Vector) to avoid fluorescence decay and nuclear staining. The plates were kept at 4°C in the dark prior to observation. In each well, three to five micrographs with random fields were acquired using a fluorescence microscope (Keyence, Osaka, Japan). The FITC‐UEA‐1 and DiI‐acLDL double‐positive cells were counted as early EPC.
3
Cytotoxicity Assay of Compounds in HSC-2 Cells
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HSC-2 cells (2 × 105 cells/2 mL/well) were seeded onto coverslips in a 6-well tissue culture plate and cultured in MEM containing 10% FBS, penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 °C under 5% CO2 atmosphere. After 24 h incubation, the medium was replaced with 2 mL fresh medium per well containing the test sample; then, the cells were cultured for 48 h. Next, the medium was removed and washed twice with PBS(–) and fixed with 4% paraformaldehyde phosphate buffer solution (pH 7.4, Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were permeabilized by 0.2% Triton X-100 in PBS(–), stained with DAPI (1 µg/mL in PBS(–)), and were observed by fluorescence microscopy (EVOS® FL Cell Imaging System, Thermo Fisher Scientific, Waltham, MA, USA) [14 (link)]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). Camptothecin was used as a reference compound.
4
Ileal Tissue Isolation and Analysis
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The mice were anesthetized using 2% isoflurane and sacrificed 1 h after the final OVA challenge on day 39, followed by isolation of ileal tissues. For ex vivo experiments using the Ussing chamber, the tissues were immediately immersed in ice-cold oxygenated Krebs buffer containing the following (mmol/L):115.0 NaCl, 1.25 CaCl2, 1.20 MgCl2, 2.0 KH2PO4, 25.0 NaHCO3, and 10.0 C6H12O6 (pH 7.35). Tissue segments were immediately frozen on dry ice and stored at −80 °C for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and protein analysis. For histological evaluation and immunofluorescence staining, the tissues were fixed with 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan.) and embedded in paraffin.
5
Tissue Histology and Immunohistochemistry Protocol
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Tissue samples were retrieved at various time points and prepared for histological and immunohistochemical examination. The specimens were fixed overnight in 4% (v/v) Paraformaldehyde Phosphate Buffer Solution (Wako, Japan), embedded in paraffin and then cross-sectioned longitudinally into 5-μm sections using a Leica microtome for Gill’s 3 hematoxylin and aqueous eosin Y solution (H&E) to visualize the overall tissue morphology. All samples were analyzed using an upright microscope, and images were acquired using ScanScope camera (Leica Biosystems)19 (link).
6
Bovine Biglycan Effect on Cell Morphology
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A total of 1 × 104 LF cells per well were incubated in 4-well plates (LAB-TEK, chamber slide w/Cover RS Glass Slide Sterile, Fisher Scientific) overnight for attachment to well surface. Thereafter, cells were stimulated with different concentrations (0, 1, 5, and 10 µg/mL) of BGN in DMEM with 10% FBS in a 5% CO2 humidified incubator. After 24-h stimulation, the cells were fixed in 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation, Chuo-Ku, Osaka, Japan) for 2 min and stained with Gram easy liquid neo-B & MP Crystal Violet Solution (FUJIFILM Wako Pure Chemical Corporation) for 2 min57 (link). Images were captured for each BGN concentration and control wells using a digital image analyzer (Model ECLIPSE TS100; Nikon, Japan). The ratio of cell length to width was measured for 14 cells57 (link), which were selected from each power field for calculation using computer software (Image-J ver. 1.51).
7
Ertredin Derivatives Inhibit Tumor Growth
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NIH3T3/EGFRvIII (1 × 105) cells or NIH3T3/EGFRwt (1 × 106) cells were suspended in 100 μL of 62.5 % v/v Matrigel (BD Biosciences) in DMEM and transplanted subcutaneously into female Balb/c nude mice at 7–8 weeks of age. Either Ertredin derivatives or gefitinib were dissolved in a vehicle consisting of saline, 10 % w/w DMSO, and 1 % w/w Tween80. After cell transplantation, 30 mg/kg Ertredin or I-Ertredin was injected intraperitoneally once daily from day 1–17. Gefitinib at 100 or 200 mg/kg was orally administrated to mice transplanted with these cells as a positive control.
Five to 6 mice were included in the vehicle-injected group, 4–5 in the Ertredin-or I-Ertredin-injected group, and 3–5 in the gefitinib-injected group. We measured the long and short diameters of the tumors every 2 or 3 days, and calculated tumor volume with the following equation: tumor volume = (long diameter) × (short diameter)2 / 2. On day 17 or 18, the tumors were extirpated, weighed, and photographed using a Nikon COOLPIX 990. The images were processed by Preview (MacOS). For TUNEL staining, parts of the tumors were fixed using 4 % paraformaldehyde phosphate buffer solution (Wako), followed by paraffin embedding.
Five to 6 mice were included in the vehicle-injected group, 4–5 in the Ertredin-or I-Ertredin-injected group, and 3–5 in the gefitinib-injected group. We measured the long and short diameters of the tumors every 2 or 3 days, and calculated tumor volume with the following equation: tumor volume = (long diameter) × (short diameter)2 / 2. On day 17 or 18, the tumors were extirpated, weighed, and photographed using a Nikon COOLPIX 990. The images were processed by Preview (MacOS). For TUNEL staining, parts of the tumors were fixed using 4 % paraformaldehyde phosphate buffer solution (Wako), followed by paraffin embedding.
8
Histological analysis of adipose tissue
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Epididymal adipose tissue was collected from WT and Plap-1 KO mice after 16 weeks of feeding HFD and then fixed in 4% Paraformaldehyde Phosphate Buffer Solution (Wako Pure Chemical Industries, Osaka, Japan) overnight. Samples were embedded in paraffin and sectioned at 3.0 μm with LEICA RM2245 (Leica Microsystems, Wetzlar, Germany). Sections were stained with Mayer’s Hematoxylin (MUTO PURE CHEMICALS, Tokyo, Japan) and 1% Eosin Y Solution (Wako Pure Chemical Industries, Osaka, Japan). Stained sections were observed and imaged with ECLIPSE Ci (Nikon, Tokyo, Japan). The size of adipocytes was measured with ImageJ software.
9
Immunofluorescence Staining of METTL6 in SNU-423 Cells
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Cells were seeded at a density of 1×104 cells/well (SNU-423_KO2) with and without Dox treatment in 2-well chamber culture slides (#154852, Lab-Tek™ II CC2™ Chamber Slide System, Thermo Fisher Scientific, Inc.). After 4 days, the cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde (#163-20145, Paraformaldehyde Phosphate Buffer Solution, FUJIFILM Wako Pure Chemical Corporation) for 15 min at 25°C. Cells were then blocked with 3% BSA (#A-9647, bovine serum albumin, Sigma-Aldrich; Merck KGaA) and 0.3% Triton X-100 [#160-24751, Polyoxyethylene(10 (link)) Octylphenyl Ether, FUJIFILM Wako Pure Chemical Corporation] for 60 min. The cells were subjected to immunofluorescence staining with rabbit polyclonal METTL6 primary antibody (#HPA035166, Sigma-Aldrich; Merck KGaA; dilution used: 1:200) overnight at 4°C. Next, the cells were washed with cold PBS three times for 5 min each time, and incubated with donkey anti-rabbit IgG (H+L) secondary antibody Alexa Fluor 594 (#A21207, Thermo Fisher Scientific, Inc., dilution used: 1:500) at 25°C for 1 h. The cells were washed with cold PBS and mounted using mounting medium containing DAPI (#H-1200, Vector Laboratories). The cells were examined using fluorescence microscopy (Celldiscover 7, ZEISS) at 20× with 5×5 tiling.
10
Multiparametric Flow Cytometry of Tumor Immune Cells
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Single-cell suspensions were obtained from mouse tumors after tumor disaggregation as described in the previous method. The cells were treated with anti-CD16/CD32 antibodies to block Fc receptors. Subsequently, the cells were stained with the following antibodies (BioLegend): Brilliant Violet 510-conjugated anti-CD45, FITC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD8a, FITC-conjugated anti-CD8a, PerCP-conjugated anti-PD-1, APC- conjugated anti-CD44, Pacific Blue-conjugated anti-CD69, PE-conjugated anti-KLRG-1, PerCP-conjugated anti-CD25, APC- conjugated anti-CD103, APC-Cy7-conjugated anti-CD4, Pacific Blue-conjugated anti-FOXP3, PerCP-conjugated anti-MHC-II, APC-Cy7-conjugated anti-CD11c, Pacific Blue-conjugated anti-CD11b, PE-conjugated anti-XCR-1. The cells were stained for 30 min at 4 °C in the dark. For intracellular staining, cells were fixed using 4% Paraformaldehyde Phosphate Buffer Solution (Wako, Osaka, Japan) and permeabilized using 0.5% Polyoxyethylene (10) Octylphenyl Ether (Wako, Osaka, Japan) then stained with antibody for 30 min at 4 °C in the dark. After extensive washing with FACS buffer, the cells were subjected to flow cytometry Canto II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (BD Biosciences, version 10.6).
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